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Biomedical subjects

Joseph Itskovitz-Eldor

Publications and source records attributed to Joseph Itskovitz-Eldor.

At least 19 recordsLinked to original sources
PubMed2007

Automated maintenance of embryonic stem cell cultures.

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

Animals↗
PubMed2006

Neural conversion of human embryonic stem cell colonies in the presence of fibroblast growth factor-2.

Pluripotency and the capability for unlimited self-renewal make human embryonic stem cells a promising tool for studying development and new cell replacement strategies. Here, we present a simple differentiation protocol, which permits the direct conversion of human embryonic stem cells into neurogenic precursors without formation of embryoid bodies or coculture with other cell types. In this protocol, human embryonic stem cells propagated as adherent cultures are induced to differentiate into the neural lineage in media containing fibroblast growth factor-2. The adherent cells are proliferated to form detaching neurospheres. Upon plating, these neurospheres give rise to a homogenous population of neural precursors capable of generating neurons, astrocytes and oligodendrocytes. Our findings suggest that fibroblast growth factor-2 exposure alone suffices to promote neural conversion of adherently growing human embryonic stem cell cultures.

Astrocytes↗
PubMed2006

Transduction of human embryonic stem cells by ecotropic retroviral vectors.

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

Animals↗
PubMed2006

Generation and characterization of functional cardiomyocytes from rhesus monkey embryonic stem cells.

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre-clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and alpha-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and beta-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre-clinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.

Animals↗
PubMed2006

Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions.

The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.

Animals↗
PubMed2006

Derivation and maintenance of human embryonic stem cells.

Since their derivation in 1998, human embryonic stem cells (hESCs) have been the center of tremendous scientific efforts in improve the existing methodologies for their isolation and maintenance to exhaust the potential use of these unique cells in cell-based therapy and developmental research. To date, there are more than 50 reported well-characterized hESC lines worldwide. hESCs are traditionally isolated from the blastocysts on mouse embryonic fibroblasts. The most used method for isolating the inner cell mass from the human blastocyst is immunosurgery. This chapter focuses on the basic methods for the derivation and maintenance of hESC lines.

Animals↗
PubMed2006

Human embryonic stem cells for vascular development and repair.

Embryonic stem cells, derived from the inner cell mass of embryos in the blastocyst stage, are cells capable of perpetual self-renewal and long-term propagation and hold the potential to differentiate to progeny of the three embryonic germ layers. Since their derivation approximately two decades ago, exploration of mouse ES cells made major advances in ES cell differentiation research and in the successful development and propagation of various cell types. The subsequent derivation of ES cells from human embryos allows detailed study of early developmental events practically unreachable in early human embryos, and the potential derivation of a variety of adult cell types differentiated from the ES cells holds immense therapeutic promise. Recently, the study of ES cell-derived teratomas identified the partial presence of human ES cell-derived premature vessels within the teratoma, and a preliminary protocol for the in vitro derivation of a vascular progenitor was developed based on the study with the mouse ES cells. Furthermore, genetic profiling identified a pattern of expression of various endothelial and vascular smooth muscle cell genes that provide additional information on the degree of vascular development that ES cells undergo. Finally, the clinical application of ES cells in transplantation medicine is closer than ever following the affirmation that human ES cell-derived endothelial progenitors conferred increased neovascularization in transplanted engineered skeletal muscle. This review summarizes these recent advances in vascular development from human ES cells and their potential clinical applications.

Animals↗
PubMed2005

Episiotomy: beliefs, practice and the impact of educational intervention.

OBJECTIVE: To assess the potential effect of educational intervention on episiotomy practice. STUDY DESIGN: A survey of the attitude of obstetrical caregivers toward episiotomy was conducted among obstetricians and midwives in the three public hospitals in Haifa. Data regarding episiotomy rates was collected for the years 2001-2003. At the beginning of 2002, lectures on the risks and benefits of episiotomy were given in two hospitals. Episiotomy rates before and after the lecture were compared. RESULTS: A significant and lasting reduction in episiotomy rates was observed in the two hospitals where lectures were given. There were no clinically significant and consistent changes in the episiotomy practices in the third hospital. CONCLUSION: Education may play an important role in changing common medical practices, as in episiotomy. It was clearly shown that our beliefs are not always up to date. We call for periodic reassessment of all medical procedures, as common and accepted as they are.

Attitude of Health Personnel↗
PubMed2005

Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction.

Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.

Animals↗
PubMed2005

Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cells.

The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large-scale gene screening. Two main clusters were identified-one of down-regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma-derived growth factor-1), LIN28, CD24, TERF1 (telomeric repeat binding factor-1), LEFTB (left-right determination, factor B), and a second of up-regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up-regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGFbeta3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGFbetaR2, and TGFbetaR3, other markers such as CD34, VCAM1, PECAM1, VE-CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development.

Antigens, CD34↗
PubMed2005

Nucleofection of human embryonic stem cells.

Human embryonic stem (hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein (EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates >70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency- associated markers Tra-1-60, Tra-1-81, and Oct4 and could be expanded to stably transgene-expressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening (HTS) applications.

Animals↗
PubMed2005

Molecular analysis of cardiomyocytes derived from human embryonic stem cells.

During early embryogenesis, the cardiovascular system is the first system to be established and is initiated by a process involving the hypoblastic cells of the primitive endoderm. Human embryonic stem (hES) cells provide a model to investigate the early developmental stages of this system. When removed from their feeder layer, hESC create embryoid bodies (EB) which, when plated, develop areas of beating cells in 21.5% of the EB. These spontaneously contracting cells were demonstrated using histology, immunostaining and reverse transcription-polymerase chain reaction (RT-PCR), to possess morphological and molecular characteristics consistent with cardiomyocytic phenotypes. In addition, the expression pattern of specific cardiomyocytic genes in human EB (hEB) was demonstrated and analyzed for the first time. GATA-4 is the first gene to be expressed in 6-day-old EB. Alpha cardiac actin and atrial natriuretic factor are expressed in older hEB at 10 and 20 days, respectively. Light chain ventricular myosin (MLC-2V) was expressed only in EB with beating areas and its expression increased with time. Alpha heavy chain myosin (alpha-MHC) expression declined in the pulsating hEB with time, in contrast to events in EB derived from mice. We conclude that human embryonic stem cells can provide a useful tool for research on embryogenesis in general and cardiovascular development in particular.

Biomarkers↗
PubMed2005

Functional properties of human embryonic stem cell-derived cardiomyocytes.

Regeneration of the diseased myocardium by cardiac cell transplantation is an attractive therapeutic modality. Yet, because the transplanted cardiomyocytes should functionally integrate within the diseased myocardium, it is preferable that their properties resemble those of the host. To determine the functional adaptability of human embryonic stem cell-derived cardiomyocytes (hESC-CM) to the host myocardium, the authors investigated the excitation-contraction (E-C) coupling and the responsiveness to common physiological stimuli. The main findings are: (1) hESC-CM readily respond to electrical pacing and generate corresponding [Ca(2+)](i) transients (measured by fura-2 fluorescence) and contractions (measured by video edge detector). (2) In contrast to the mature myocardium, hESC-CM display negative force-frequency relations. (3) The hESC-CM contraction is dependent on [Ca(2+)](o) and blocked by verapamil. (4) Surprisingly, ryanodine, the sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, and caffeine do not affect the [Ca(2+)](i) transient or contraction. Collectively, these results indicate that at the developmental stage of 45 to 60 days, the contraction is largely dependent on [Ca(2+)](o) rather than on sarcoplasmic reticulum (SR) Ca(2+) stores. The results show for the first time that the E-C coupling properties of hESC-CM differ from the adult myocardium, probably due to immature SR function. Based on these findings, genetic manipulation of hESC-CM toward the adult myocardium should be considered.

Animals↗
PubMed2005

High-efficiency RNA interference in human embryonic stem cells.

RNA interference methodology suppresses gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. In this study, we used retroviral and lentiviral vectors to deliver small interfering RNAs and report high-efficiency silencing of a green fluorescent protein (GFP) trans gene and the stem cell-specific transcription factors Oct4/POU5F1 and Nanog in human embryonic stem cells. Gene knockdown of Oct4 and Nanog promotes differentiation, thereby demonstrating a role for these factors in human embryonic stem cell self-renewal.

Antigens, Surface↗
PubMed2005

CD133-positive hematopoietic stem cell "stemness" genes contain many genes mutated or abnormally expressed in leukemia.

Affymetrix human Hu133A oligonucleotide arrays were used to study the expression profile of CD133+ cord blood (CB) and peripheral blood (PB) using CD133 cell-surface marker. An unsupervised hierarchical clustering of 14,025 valid probe sets showed a clear distinction between the CD133+ cells representing the hematopoietic stem cell (HSC) population and CD133-differentiated cells. Two hundred forty-four genes were found to be upregulated by at least twofold in the CD133-positive cells of both CB and PB compared with the CD133-negative cells. These genes represent the hematopoietic "stemness," whereas the 218 and 304 upregulated genes exclusively in PB and CB, respectively, represent tissue specificity. Some of the stemness genes were also common to HSC genes found to be upregulated in several recently published studies. Among these common stemness genes, we identified several groups of genes that have an important role in hematopoiesis: growth factor receptors, transcription factors, genes that have an important role in development, and genes involved in cell growth. Sixteen selected stemness genes are known to be mutated or abnormally regulated in acute leukemias. It can be suggested that key hematopoietic stemness machinery genes may lead to abnormal proliferation and leukemia upon mutation or change of their expression.

AC133 Antigen↗
PubMed2005

[Insemination from HIV-positive males].

INTRODUCTION: Discordant couples, in which the man is HIV positive and the woman is HIV negative, face limited options when they wish to produce healthy children whilst practicing safe sex. In order to conceive they must abandon unprotected sex, which carries high risk of HIV infection to the woman. OBJECTIVE: To develop a method for removing the HIV from semen and then perform intra uterine insemination, following verification of the HIV-negative spermatozoa fraction by PCR. METHODS: Motile spermatozoa were isolated from semen samples by the gradient and "swim-up" techniques. HIV-RNA was tested before and after the procedure in both the semen and the purified spermatozoa fraction. Insemination was performed on ovulation day only when the absence of any viral particles was verified by PCR. RESULTS: Four couples underwent a total of 8 cycles of intra uterine insemination (IUI). Presently, two healthy babies were born. The two mothers and newborn were found to be HIV negative following the delivery. CONCLUSION: We report a safe procedure which enables biological parenthood for HIV-1 discordant couples without the risk of infecting the female partner. Our clinical data corroborate with the collaborative European data on this subject.

Adult↗